Assay and reagents for quantifying hBNp

Abstract

The present invention provides reagents and assays for the quantification of hBNP in biological fluid samples such as plasma or serum. Antibodies are provided which are monospecific to epitopes comprising the amino acid sequences 5-13, 1-10 and 15-25 of hBNP. These antibodies, and peptide fragments containing these sequences, can be employed in the assays of the invention, which may be carried out in a sandwich format or a competition format.

What is claimed is:  
     
       1. A method of quantifying hBNP in a biological fluid which method comprises forming a labeled complex said complex consisting of 
       (a) a first antibody selected from the group consisting of:  
       (i) an antibody that is monospecifically reactive to an hBNP fragment hBNP 5-13 (positions 2-10 of SEQ. ID. NO: 2) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 5-13;  
       (ii) an antibody that is monospecifically reactive to an hBNP fragment hBNP 1-10 (positions 1-10 of SEQ. ID. NO: 3) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 1-10;  
       (iii) an antibody that is monospecifically reactive to an hBNP fragment hBNP 15-25 (positions 2-12 of SEQ. ID. NO: 4) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 15-25;  
       (b) any hBNP contained in said biological fluid;  
       (c) a second antibody immunoreactive with hBNP or a fragment of said antibody that is immunoreactive with hBNP, and  
       (d) at least one label; and  
       determining the amount of hBNP in the sample by quantifying the label in said complex.  
     
     
       2. The method of  claim 1  comprising the step of separating a first antibody-hBNP-second antibody complex formed by the combination of (a), (b) and (c) of  claim 1  from the remainder of the biological fluid sample prior to quantifying the label in the said labeled complex and either before or after said complex is labeled. 
     
     
       3. The method of  claim 2  wherein separation of the first antibody-hBNP-second antibody complex from the remainder of the biological fluid sample is effected by binding the first or second antibody to a solid support and removing the remainder of the biological fluid sample from contact with said solid support after formation of the first antibody-hBNP-second antibody complex. 
     
     
       4. The method of  claim 1  wherein the label is selected from the group consisting of radioactive labels, enzymatic labels, fluorescent labels, thermometric labels and immunopolymerase chain reaction labels. 
     
     
       5. The method of  claim 4  wherein the label is an enzymatic label. 
     
     
       6. The method of  claim 1  wherein a first antibody-hBNP-second antibody complex formed by the combination of (a), (b) and (c) is labeled after formation thereof by contacting said complex with a labeled antibody that binds to the first or second antibody without displacing said first or second antibody from the complex. 
     
     
       7. The method of  claim 1  wherein the biological fluid is plasma. 
     
     
       8. The method of  claim 1  wherein the first antibody is a monoclonal antibody. 
     
     
       9. The method of  claim 1  wherein the first antibody is a monoclonal antibody that is monospecifically reactive to an hBNP fragment hBNP 5-13 (positions 2-10 of SEQ. ID. NO: 2) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 5-13. 
     
     
       10. The method of  claim 1  wherein the first antibody is a monoclonal antibody that is monospecifically reactive to an hBNP fragment hBNP 1-10 (positions 1-10 of SEQ. ID. NO: 3) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 1-10. 
     
     
       11. The method of  claim 1  wherein the first antibody is a monoclonal antibody that is monospecifically reactive to an hBNP fragment hBNP 15-25 (positions 2-12 of SEQ. ID. NO: 4) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 15-25. 
     
     
       12. A method of quantifying the amount of hBNP in a biological fluid which comprises: 
       contacting a sample of the biological fluid with a combination of reagents selected from the group consisting of:  
       (a) the combination of  
       (i) an antibody that is monospecifically reactive to an hBNP fragment hBNP 5-13 (positions 2-10 of SEQ. ID. NO: 2) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 5-13, and  
       (ii) hBNP or a fragment thereof comprising amino acids 5-13 of hBNP, having bound thereto a quantifiable label,  
       (b) the combination of  
       (i) an antibody that is monospecifically reactive to an hBNP fragment hBNP 1-10 (positions 1-10 of SEQ. ID. NO: 3) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment hBNP 1-10; and  
       (ii) hBNP or a fragment thereof comprising amino acids 1-10 of hBNP, having bound thereto a quantifiable label,  
       (c) the combination of  
       (i) an antibody that is monospecifically reactive to an hBNP fragment hBNP 15-25 (positions 2-12 of SEQ. ID. NO: 4) or a fragment of said antibody that is monospecifically reactive to said hBNP fragment h]BNP 15-25; and  
       (ii) hBNP or a fragment thereof comprising amino acids 15-25 of hBNP, having bound thereto a quantifiable label,  
       under conditions that allow the formation of an antibody-hBNP complex; and  
       determining the amount of hBNP in the sample by quantifying the label in the antibody-hBNP complex.  
     
     
       13. The method of  claim 12  further comprising the step of separating the antibody-hBNP complex from the remainder of the biological fluid sample prior to quantifying the label in the antibody-hBNP complex. 
     
     
       14. The method of  claim 13  wherein separation of the antibody-hBNP complex from the remainder of the biological fluid sample is effected by binding the antibody to a solid support and removing the biological fluid sample from contact with the solid support after formation of the antibody-hBNP complex. 
     
     
       15. The method of  claim 12  wherein the antibody is a monoclonal antibody. 
     
     
       16. The method of  claim 12  wherein the label is selected from the group consisting of radioactive labels, enzymatic labels, fluorescent labels, thermometric labels and immunopolymerase chain reaction labels. 
     
     
       17. The method of  claim 12  wherein the biological fluid is plasma. 
     
     
       18. The method of  claim 12  wherein said combination is the combination of (a). 
     
     
       19. The method of  claim 12  wherein said combination is the combination of (b). 
     
     
       20. The method of  claim 12  wherein said combination is the combination of (c).